Fig. 5. Inhibition of ERK1/2 prevents translocation of pre-TNF to the cell membrane. RAW cells were plated on Labtek chambers and then pre-treated for 60 minutes with 10 µM of the TACE inhibitor TAPI-1 (A-F), with (C,F) or without (A,B,D,E) 2 µM PD 184352, and were then either left untreated (A,D) or were stimulated for 4 hours with 100 ng/ml LPS (B,C,E,F). Cells were either left unpermeabilised (A-C) or were permeabilised with 0.1% (w/v) saponin (D-F). TNF was visualised using an antibody coupled to Alexa-Fluor-488-conjugated anti-sheep IgG by confocal microscopy at 600-fold magnification. Similar results were obtained in three independent experiments.